Factor regulating gene expression

ABSTRACT

A recombinant DNA molecule coding for a protein having the activity of an interferon regulatory factor-1 (IRF-1).

This application is a division of application Ser. No. 08/347,251, filedNov. 18, 1994, (status: U.S. Pat. No. 5,616,698) which is a continuationof application Ser. No. 08/087,465, filed on Jul. 8, 1993, (status:abandoned), which is a continuation of application Ser. No. 07/397,967,filed Aug. 24, 1989, (now abandoned) originally entitled FactorRegulating Gene Expression.

FIELD OF THE INVENTION

The present invention relates generally to the regulation of geneexpression. In particular the invention relates to a recombinant DNAmolecule coding for a protein having the activity of an interferonregulatory factor-1 (IRF-1); to recombinant DNA molecules characterizedby a DNA sequence coding for an IRF-1 active protein and a promoter andregulator sequence operably linked thereto; to the use of such DNAmolecules for transforming host cells which are also transformed by DNAmolecules coding for a desired protein and under the control of saidIRF-1 active protein; to such DNA molecules including a sequence codingfor a pharmaceutically active protein and a promoter region of the genefor said protein including a binding site for the IRF-1 active molecule;and to the production of said IRF-1 active protein and/or saidpharmaceutically active protein by cultivation of suitable host cellstransformed by said DNA molecules.

BACKGROUND OF THE INVENTION

Transcription of genes in mammalian cells is regulated by complexmechanisms wherein interactions of the regulatory DNA sequences withtrans-acting DNA binding proteins play a central role. In the context ofthe regulation of transcription, genes encoding interferons (IFNs)represent a feature common to many of the cytokine genes; transcriptionof those genes is induced in a transient manner following variousextra-cellular signals. It has been well documented that transcriptionof the genes for IFN-α and IFN-β is efficiently induced by viruses in avariety of cells, while that of the gene encoding IFN-γ is induced in Tlymphocytes (T cells) following mitogenic stimulation (for a review seeWeissmann and Weber, 1986; Taniguchi, 1938). IFN-β, a cytokine that wasoriginally identified for its potent antiviral activity, also appears toplay a crucial role in controlling cell growth and differentiation. Inthis regard, beside viruses and poly(rI):poly(rC) which are the wellknown inducers of IFN-β gene, many of the cytokines such as colonystimulating factor-1 (CSF-1) (Moore et al., 1984; Warren and Ralf, 1986;Resnitzky et al., 1986), tumor necrosis factor (TNF) (Onozaki et al.,1988), platelet-derived growth factor (PDGF) (Zullo et al., 1985) andIFNs (Kohase et al., 1987) also appear to induce IFN-β in certain cells,suggesting that they may transduce similar or identical signals in thetarget cells.

The IFN-β gene induction by viruses and poly(rI):poly(rC) has been shownto occur at the transcriptional level (Raji and Pitha, 1983; Ohno andTaniguchi, 1983; Dinter et al., 1983; Zinn et al., 1983). Thuscis-acting DNA sequences functioning as inducible enhancers have beenidentified within the 5'-flanking region of the human IFN-β gene (Fujitaet al., 1985, 1987; Goodbourn et al., 1985; Dinter and Hauser, 1987).The inducible enhancer region (i.e. -65 to -105 with respect to the CAPsite) contains repetitive hexanucleotide units some of which indeedfunction in the induced-activation of transcription when multimerized(Fujita et al., 1987). We have identified in mammalian cells such asmouse L929 cells and human cells, a factor, IRF-1, which specificallybinds to the IFN-β regulatory sequences, as well as to the functional,repeated hexanucleotide sequences; (AAGTGA)₄. We have found that IRF-1plays an essential role in virus-induced activation of IFN-β genetranscription by interacting with the identified cis-elements.

SUMMARY OF THE INVENTION

According to a broad aspect of this invention we provide a recombinantDNA molecule coding for a protein having the activity of an interferonregulatory factor-1 (IRF-1).

Preferably the DNA molecule codes for or is hybridizable to the DNAmolecule coding for human IRF-1 or mouse (murine) IRF-1.

The DNA molecule is preferably one which codes for a protein which bindsto the repeated oligomer sequence AAGTGA and the regulatory upstreamelements of the human IFN-β-gene.

The DNA molecule may include a promoter region which is constitutive orinducible, for instance virus inducible e.g. by Newcastle Disease Virusor is inducible by mitogenic stimulation e.g. using Concanavalin A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Gel Retardation Assay.

FIG. 2: DNAase Footprinting Analysis. The sequence on the left side ofFIG. 2 is part of a DNA sequence of the wild type IFN-β probe. The DNAsequence on the right side of FIG. 2 is part of the DNA sequence of themutant IFN-β probe.

FIG. 3: DNA Competition Assay: the left hand panel shows the resultsobtained when the hexamers were used as competitor; the middle panelgives the results when human EFN gene segments were used as competitors;the right hand panel gives the results when the various DNA segments asindicated within the panel were used.

FIGS. 4A-4C: The DNA and deduced protein sequence of the cDNA insert ofpIRF-L; FIG. 4D: Hydropathy plot analysis.

FIG. 5: Comparison of the deduced animo acid sequences of murine andhuman IRF-1. The conserved amino acids are marked by asterisks. Thesequences are presented with the one letter amino acid code as follows:A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F,phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L,leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R,arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y,tyrosine.

FIG. 6: Expression of IRF-1 mRNA: (6A) Analysis of different tissues;(6B) Analysis as a function of time with IRF-1, IFN-β, and β-actin.

FIGS. 7A-7B: (7A) Nucleotide sequence of the PstI fragment from Ig14-2which contains the mouse IRF-1 promoter sequence; (7B) Relative CATactivity.

FIG. 8: Nucleotide and deduced amino acid sequence of the human IRF-1gene.

FIG. 9: Identification of DNA sequences present in yeast thatcross-hybridize with human IRF-1 cDNA.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

One preferred DNA molecule (coding for human IRF-1) is characterised bya structural gene having the formula I below: ##STR1## or a dengeneratevariant thereof.

The DNA molecule may for example be characterized by a structural genehaving the formula defined above and upstream and downstream flankingsequences contained within the following formula II: ##STR2## or adegenerate variant thereof.

Another preferred DNA molecule (coding for murine IRF-1) ischaracterized by a structural gene having the following formula III:##STR3## or a degenerate variant thereof.

Such a DNA molecule as mentioned in the foregoing paragraph may, forexample, be characterized by a structural gene having the formuladefined above and upstream and downstream flanking sequences containedwithin the following formula IV: ##STR4## or a degenerate variantthereof.

DNA sequences of the invention coding for proteins having IRF-1 activityinclude DNA sequences from a eukaryotic source such as, not only humanand mouse but also, e.g. chicken, frog and yeast, which hybridize withthe DNA sequences of the foregoing human or murine IRF-1 (FIGS. I to IV)and which code for a protein having IRF-1 activity.

One such cDNA molecule which hybridize to the cDNA of murine IRF-1 asdefined in FIG. 4A (and is named IRF-2) is set forth below in formulaIIIa: ##STR5## or is a degenerate variant thereof.

This DNA molecule may, for example be characterized by a structural genehaving the formula defined above and upstream and downstream flankingsequences contained within the following formula IVa: ##STR6## or adegenerate variant thereof.

We describe below the isolation of cDNA molecules from human cells andmouse cells coding for human and murine IRF-1 and from mouse cells andyeast which hybridize respectively to the cDNA of murine IRF-1 and humanIRF-1.

The recombinant DNA molecule may also contain a promoter and regulatorysequence contained within the following formula V: ##STR7## or adegenerate variant thereof.

The recombinant DNA molecule may also be designed for expression of apharmaceutically active protein such as e.g. a cytokine or a plasminogenactivator and in this form will contain preferably a structural gene fora desired pharmaceutically active protein operably linked to a promoterregion of the gene for the said protein including a binding site for theIRF-1 active molecule.

Thus a recombinant DNA molecule of the invention may comprise a DNAsequence as defined above and a structural gene for a desiredpharmaceutically active protein under the control of the IRF-1 activeprotein coded for by said DNA sequence. In such a recombinant DNAmolecule the gene coding for the IRF-1 active molecule is preferablyunder the control of a constitutive promoter or most preferably aninducible promoter. Preferably the gene for the desired pharmaceuticallyactive protein will include an IRF binding site containing repetitiveAAGTGA sequences.

The present invention also comprehends a host cell e.g. a bacterial celle.g. E. coli, or a yeast cell or a mammalian cell e.g. a CHO cell or amouse cell e.g. L929, transformed by a recombinant DNA molecule asdefined above. Ideally the host cell will be selected from a cell linewhich has no or substantially no level of endogenous IRF-1 activity.

Alternatively for the production of a pharmaceutically active protein ahost cell may be transformed by a first DNA molecule containing asequence coding for a protein having the activity of an IRS-1, and by asecond separate DNA molecule containing a gene coding for a desired,pharmaceutically active protein that is under the control of the IRF-1active protein that is coded for by the first DNA molecule. Preferablythe first DNA molecule coding for the IRF-1 active molecule includes aconstitutive promoter or most preferably an inducible promoter sequenceoperably linked to the gene coding for the IRF-1 active compound. Also,preferably the second DNA molecule includes a binding site for the IRF-1active protein containing repetitive AAGTGA sequences.

The IRF-1 active protein or the pharmaceutically active protein can beproduced by cultivation of the transformed cell and isolation of theproduced protein in conventional manner.

Suitably the host cells are induced by treatment in a manner appropriateto the promoter which is operably linked to the gene coding for theIRF-1, as discussed below.

The invention also comprehends a protein having the activity of an IRF-1obtained by the cultivation of a host transformed with a DNA molecule asdefined above.

A preferred protein having the activity of an IRF-1 has the formula VI:

    __________________________________________________________________________    Formula VI                                                                    __________________________________________________________________________    Met                                                                              Pro                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Met                                                                              Arg                                                                              Met                                                                              Arg                                                                              Pro                               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Another preferred protein having the activity of an IRF-1 has theformula VII: ##STR8##

Yet another preferred protein (IRF-2), which is coded for by the cDNAsequence of formula IIIa has the formula VIII: ##STR9##

We describe below the molecular cloning and characterization of murineand human cDNA encoding DNA binding proteins having the IRF-1 activity.

A remarkable sequence conservation will be seen between the murine andhuman IRF-1 molecules, as revealed from the analysis of the clonedcDNAs. Furthermore, expression of the gene encoding IRF-1 is shown to beinduced by Newcastle Disease Virus (NDV) and Concanavalin A (ConA) inmouse L929 cells and splenic lymphocytes, respectively

As used herein, a "functional derivative" is a compound which possessesa biological activity (either functional or structural) that issubstantially similar to a biological activity of IFR-1. The term"functional derivative" is intended to include the "fragments,""variants," "analogues," or "chemical derivatives" of a molecule. A"fragment" of a molecule such as IFR-1, is meant to refer to anypolypeptide subset of the molecule. A "variant" of a molecule such asIFR-1 is meant to refer to a molecule substantially similar in structureand function to either the entire molecule, or to a fragment thereof. Amolecule is said to be "substantially similar" to another molecule ifboth molecules have substantially similar structures or if bothmolecules possess a similar biological activity. Thus, provided that twomolecules possess a similar activity, they are considered variants asthat term is used herein even if the structure of one of the moleculesis not found in the other, or if the sequence of amino acid residues isnot identical.

EXAMPLES

1. Cloning and expression of IRF-1 DNA in E. coli

A. Poly(A)⁺ RNA was isolated from uninduced mouse L929 cells and used tosynthesis cDNA (following the procedure of Aruffo and Seed, 1987). Theresulting cDNA was then cloned into an EcoRI-cleaved λgt11 vector and acDNA library constructed according to the standard procedure of Huynh etal., 1985, using E. coli Y1090 as the host strain.

The resulting λgt11 library was then screened using the multimerized (4times) AAGTGA sequence (hereinafter referred to as the C1 oligomer;Fujita et al., 1987) as the probe.

In this screening procedure E. coli Y1090, infected by the recombinantλgt11 phages was plated onto 10×13 cm square plates. The plates werethen incubated at 12° C. for 4 to 5 hours when about 20,000plaques/plate were visible.

Membrane filters for screening were either nylon (Nytran; Schleicher &Schnell) or nitrocellulose (Schleicher & Schnell) membranes. The filterswere immersed in 10 mM IPTG (Isopropyl-b-D-thiogalactopyranoside for theinduction of lacZ gene expression in the appropriate phage plaques) andair dried, then overlayed on the plates and incubated at 37° C. for 2.5hours. Plaques will only produce a protein encoded by cDNA if the cDNAis in-frame with the lacZ gene.

The filters were then removed and chilled at 4° C. for 20 minutes and,without drying, were subjected to screening.

The membrane filters were then prepared for assay as follows:

Nuclear extract was prepared from mouse L929 cells and it was spotted(in an amount corresponding to about 10 μg protein) onto the membranes.

Prior to effecting DNA binding a Binding Buffer consisting of 10 mMHepes, pH 7.5, 5.0 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol was used.5% of non-fat powder milk (Yukijurishi Inc.) was added to the buffer andthe filters were incubated in the mixture for 1 hour at 4° C. and thenrinsed for 1 minute in the same buffer but containing no powder milk.

The filters were then incubated in binding buffer (1 ml) containing 350μg/ml of salmon sperm DNA of average length of approximately 300 bp and³² P-labelled probe C1 oligomer DNA (10⁶ cpm/ml; specific activity 2,000cpm/f mole) (see Fujita et al., 1987). The probe DNA was end labeled atthe 5'-termini γ-³² P!ATP using T4 kinase.

After the binding, the filters were washed at room temperature with theBinding Buffer for 1 to 2 hours (10 ml filter) changing the Bindingbuffer several times during this operation. The filters were thenair-dried and subjected to autoradiography. In this assay the nylonmembranes, but not the nitrocellulose membranes, gave a positive signalwhich was specifically inhibited by including excess unlabeled C1oligomer.

About 1.4×10⁶ recombinants were screened in this way. Among the 32positive phage clones identified in the first screening, one clone(designated λL28-8) was found to bind repeatedly with the probe DNA insubsequent rounds of screening.

2. Production and Purification of protein in transfected E. coli

Lysogenic bacterial clones were prepared by transfecting E.coli Y1089with λL28-8. Overnight lysogens harboring λL28-8 were then seeded at 1%in 400 ml L-Broth. The bacteria were grown at 31° C. until the OD₆₀₀became 1. The temperature was then shifted to 42° C. for 20 min. IPTGwas then added to 10 mM and, after incubating at 38° C. for a further 20min. the cultures were rapidly pelleted and suspended in 10 ml of LysisBuffer which consists of 20 mM Hepes pH 7.9, 0.2 mM EDTA, 0.5 mMspermidine, 0.15 mM spermine, 0.1 mM DTT, 10% glycerol, 0.5 mMphenylmethyonylsulphonylfluoride (PMSF), 1 μg/ml pepstatin A, 1 μg/mlleupeptine, 500 μM L-I-tosylamide-2-phenylethylchloromethylbenzamidine,10 mM sodium molybdate, 2 mM sodium pyrophosphate and 2 mM sodiumorthovanadate. Cell suspensions were subjected to three rapidfreeze-thaw cycles and subsequently centrifuged at 30,000 rpm for 1 hr.at 4° C. using a Beckman 50 Ti rotor. The supernatant was used eitherdirectly for a gel retardation assay (see below) or was furtherpurified.

Further purification was carried out as follows:

Approximately 4 ml of the supernatant was applied on poly(di-C):poly(di-C)-column with a bed volume of 2 ml, and equilibrated with LysisBuffer. The flow-through material (4 ml) was then applied on a DE 52(Whatman) column having a bed volume of 2 ml equilibrated with Buffer Z(25 mM Hepes, pH 7.8, 12.5 mM MgCl₂, 1 mM DTT, 20% glycerol, 0.1% NP-40(NONIDET™ P-40, an octylphenolethylene oxide condensate having anaverage of 9 moles ethylene oxide per mole of phenol), 0.5 mM PMSF). TheDNA binding activity was eluted by Buffer Z containing 0.1M KCl. Theeluate (approximately 4 ml) was further concentrated using centricon-10(Amicon). The final protein concentration was 28 mg/ml.

3. Characterisation of the Protein Product of the Clone λL28-8:

(1) Gel retardation assay

Lysogenic bacterial clones harbouring λL28-8 were prepared bytransfecting E. coli Y1089 and were induced to express the cloned cDNAat high levels using the procedure of Huynh et al., 1985. Lysogenicclones prepared and treated in the same manner with the phage lackingthe cDNA insert (designated λ6) were used as a control.

Extracts each containing 3 μg protein were prepared from the inducedcultures of the Lysogen (four preparations designated λL28-8a, λL28-8band λ6a and λ6b).

Nuclear extract (3 λg protein) was also prepared from mouse L929 cells.

The extracts were incubated with 1 fmole labeled C1 oligomer probe asdescribed above having a specific activity of 8,000 cpm/fmole.

Each extract was also subject to competitive assay in which a competitorDNA was added at various concentrations to the incubation.

The results of the assay are shown in FIG. 1 in which the various lanescorrespond to the following:

    ______________________________________                                        Lane      Extract                                                             ______________________________________                                        1         none                                                                2         λ6a                                                          3         λ6b                                                          4         λ6b with 1,000 fold molar excess of unlabeled                          C1 oligomer                                                         5         λb with 1,000 fold molar excess of unlabeled                           C5A oligomer*                                                       6         λL28-8a                                                      7         λL28-8b                                                      8         λL28-8b with 1,000 fold molar excess of un-                            labeled C1 oligomer                                                 9         L28-8b with 1,000 fold molar excess of un-                                    labeled C5A oligomer*                                               10        L929 cell                                                           11        L929 cell with 1,000 fold molar excess of un-                                 labeled C1 oligomer                                                 12        L929 cell with 1,000 fold molar excess of un-                                 labeled C5A oligomer*                                               ______________________________________                                         *The C5A oligomer is described in Fujita et al., 1985, and is a 6 times       repeated GAAA sequence.                                                  

From FIG. 1 it will be apparent that bound probes are detectable inlanes 6, 7, 9, 10 and 12.

As shown in FIG. 1 protein extracts from the λL28-8 lysogens gave riseto shifted bands (lanes 6 and 7), the appearence of which was inhibitedby excess unlabeled C1 oligomer DNA but not by the same amount of theC5A oligomer (lanes 8 and 9).

In contrast to λL28-8-derived proteins those prepared from the inducedλ6-derived lysogens failed to give such shifted bands (lanes 2-5). Theshifted bands can be seen to be closely similar to those of the naturalIRF-1 from mouse L929 cells (lanes 10 and 12). Such differences as existin the two sets of shifted bands is considered to be a consequence ofthe different amounts of protein bound to the probe DNA.

In addition, it is found that the shifted bands were detectable onlywith the proteins from IPTG-induced Y1089 cells transfected with λL28-8.

(ii) DNAase Footprinting analysis

Footprinting analysis was carried out to test the binding properties ofthe protein encoded by λL28-8 cDNA to a DNA encoding the IFN-β geneupstream region.

Protein encoded by λL28-8 cDNA was extracted from the induced lysogenand partially purified by column chromatography as described above andtested for its binding properties to a DNA containing the IFN-β geneupstream region.

Probe DNAs were prepared as SalI-HindIII fragments isolated fromp-125cat (containing the wild type IFN-β gene) and p-125DPcat(containing a mutant IFN-β gene). The plasmid p-125cat was constructedas p-105cat (Fujita et al. , 1987), except the BamHI (-125)-TagI (+19)fragment from pSE-125 (Fujita et al., Cell, Vol. 41, pp 489-496, 1985)was used. Plasmid p-125DPcat, carrying point mutations within the IFN-βregulatory elements were obtained by synthetic oligo nucleotide directedmutagenesis on p-125cat as described in Hatakeyama et al., Proc. Natl.Acad. Sci. U.S.A., Vol. 83, pp 9650-9654, 1986). Both DNAs were labeledat the HindIII site by γ-³² P!ATP using T4 kinase.

4 fmoles of the probe DNA (specific activity 3,000 cpm/fmole) wasincubated in the 20 μl reaction mixture containing 25 mM Tris-HCl, pH7.9, 6.25 mM MgCl₂, 50 mM KCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, 2%polyvinylalcohol in the presence or absence of 280 μg of the purifiedprotein.

In the assay 5×10⁻⁴ unit of DNAase I (Worthington) was added andincubated for 1 min. at 25° C.

FIG. 2 shows autoradiograms of the DNA fragments obtained from samplesobtained by carrying out the following procedures. On the left hand sideof FIG. 2 is part of the DNA sequence of the wild type IFN-β probe, andon the right hand side of FIG. 2 is part of the DNA sequence of themutant IFN-β probe.

1. The wild type IFN-β probe was cleaved by A+G reactions (see Methodsin Enzymology, Vol. 65, pp 499-560)--result shown in lane 1.

2. The wild type IFN-β probe was partially digested by DNAase I withoutprotection--result shown in lane 2.

3. The wild type IFN-β probe was reacted with the protein and thendigested with DNAase I--result shown in lane 3.

4. The wild type IFN-β probe was reacted with the protein in thepresence of 1,000 fold molar excess of unlabeled C1 oligomer and thendigested with DNAase I--result shown in lane 4.

5. The mutant IFN-β probe was reacted with the protein and then digestedby DNAase I--result shown in lane 5.

6. The mutant IFN-β probe was cleaved by A+G reactions--result shown inlane 6.

In FIG. 2 the protected regions as revealed in lanes 3 and 5 areindicated respectively on the sequences depicted on the left and rightsides of the autoradiogram. The hexamer motifs are framed.

From the results in FIG. 2 it will be seen that the protected regioncorresponds to nucleotides -100 to -64, this being the region found tobe protected by IRF-1 obtained from L929 cells. The protection wasabrogated by the use of excess unlabeled C1 oligomer (lane 4). It wasalso found, using lower protein concentrations, that preferentialprotection occurred in the region containing the AAGTGA motif (-80 to-70).

These results indicate that the protein has a higher affinity to theregion containing the AAGTGA motif and a lower affinity to thesurrounding region.

The mutant IFN-β gene segment carries T- G mutations at positions -106,-100, -73 and -67. In comparing lanes 5 and 2, under the same assayconditions the protection afforded by the recombinant proteins appearsrestricted to the unmutated region (lane 2). This observation is inconformity with the observation that the introduction of these mutationsresults in a dramatic reduction (20 fold) of the inducibility oftranscription by NDV in L929 cells and that in vitro binding of IRF-1 tothe mutant IFN-β gene was notable only in the unmutated region.

Further, the use of the C1 oligomer reveals that the proteinspecifically protects the region containing the oligomer sequences. Thisprotection corresponded identically to that afforded by native IRF-1derived from L929 cells.

3. DNA Competition Assay

This assay was carried out to examine the affinity of the recombinantprotein to various DNA sequences including some of the knowntranscriptional regulatory DNA sequences. The procedure was as follows:

The HindIII-SalI fragment of IFN-β probe was isolated from p-125 cat(see Fijita et al., 1987); this fragment contains the human IFN-β genesequence from +19 to -125. The DNA was labeled at the 3' termini byfilling in both ends with α-³² P! dCTP using Klenow fragment.

The specific activity of the probe DNA was 8,000 cpm/fmole.

The gel retardation assays were carried out under the conditionsdescribed above.

In the competition assay runs the DNA was reacted with the protein inthe presence of various concentrations of competitor DNAs in the bindingmixture as indicated in FIG. 3.

The formation level of the complex was quantitated by densitometricanalysis of the autoradiogram. Complex formation in the absence ofcompetitor DNA was taken to be 100%.

The structure of the competitor DNAs were as follows:

a) AP-1: a synthetic DNA having the following sequence ##STR10## b) TNF:37 bp of synthetic DNA that encompasses from +1162 to +2116 (see Nedwinet al., 1985) of the TNF-α first intron;

c) murine H2-D^(d) : 37 bp of synthetic DNA that encompasses the IRSelement (-159 to -123) as described by Korber et al., 1988;

d) human IFN-α: 46 bp synthetic DNA corresponding to virus ResponseElement, see Ryals et al., 1985.

e) Hexamer sequences C1, C2, C3, C4 and C5A. The sequences C1 and C5Aare as described above. Sequences C2, C3 and C4 are as published inCell, 49, 352-367 (1987); they represent the sequences

AAATGA--C2

AAGGGA--C3 and

AAAGGA--C4 respectively

f) the IFN-β gene sequence from +19 to -66.

In FIG. 3 the left hand panel shows the results obtained when thehexamer repeats were used as competitors. The middle panel gives theresults when human IFN gene segments were used as competitors, and theright hand panel gives the corresponding results, using various DNAsegments as indicated within the panel.

From the results shown in FIG. 3 it will be seen that the appearance ofthe shifted band was competed out by the hexamer sequences in order ofefficiency C1- C2- C3- C4, but was not competed out significantly byC5A.

It can also be observed that the synthetic DNA segment encompassing theregulatory elements of either human IFN-α1 or murine H-2D^(d) genes gaverise to a competitive activity. This is particularly interesting as theDNA segment of the H-2D^(d) gene contains the so-called IFN-responsesequence (IRS) that functions as an enhancer when the cells respond toIFN (Sugita et al., 1987; Israel et al., 1986; Korber et al., 1988).

In fact, sequence motifs similar or identical to these found on theIFN-β gene are found in many of the promoter sequences of theIFN-inducible genes where nuclear factors appear to bind specifically(Korber et al., 1988; Lery et al., 1988).

The results given in FIG. 3 are closely similar to those obtained whenthe assay is repeated under similar conditions using natural proteinproduced from L929 cells.

Structure of the CDNA encoding murine IRF-1

The DNA sequence of the cloned DNAs was determined either by a dideoxymethod (SEQUENASE; United States Biochemical, Inc.) or by the standardMaxam-Gilbert method (Maxam and Gilbert, 1980).

The λL28-8 insert in E.coli Y1089 was isolated as follows: the phage DNAwas prepared by standard procedure and the DNA was digested by EcoRIthen the cDNA cleaved out of the phage DNA was isolated and sequenced bythe dideoxy method (Sequenase: United States Biochemical Inc.). The cDNAinsert in λL28-8 was found to be 1.8 kb long. The nucleotide sequenceanalysis revealed a large open reading frame linked in phase with theβ-galactoside gene.

To screen clones containing larger cDNA inserts, double stranded cDNAwas synthesised with L929 cell derived poly (A)⁺ RNA and cloned intovector CDM8 according to the published procedure of Aruffo and Seed(Prod. Natl. Acad. Sci., U.S.A., Vol 84, pp 8573, 1987) and Seed(Nature, Vol. 329, pp. 840-842, 1987).

The recombinant plasmids were introduced into E.coli strain MC1061/p3(according to the procedure of Aruffo and Seed; as above) and the clonesof cDNA were screened using the λL28-8-derived (³² p-labeled) cDNA probeunder low stringent conditions for DNA-DNA hybridization (Kashima et al,Nature, Vol. 313, pp 402-404, 1985), and a clone pIRF-L was selected forfurther study.

The desired cDNA insert of pIRF-L was obtained by digestion with HindIIIand XbaI and sequenced by the methods described above. The sequence isshown in Formula IV and in FIG. 4.

The cDNA sequence from λL28-8 was found to contain an identical sequencein the overlapping region except that one A residue was missing betweennucleotides 1773 and 1781.

The 5' and 3' termini of the λL28-8 derived CDNA are marked by arrows inFIG. 4. The ATTTATTTA and ATTTA sequences which possibly confer the mRNAinstability are framed.

As will be apparent from FIG. 4A the cDNA of the murine cDNA derivedfrom pIRF-L was 198 bp and 20 bp longer than that of the λL28-8 cDNA inthe 5'and 3' regions, respectively.

Analysis of the genomic DNA sequence containing the promoter region ofthis gene reveals that the cDNA of pIRF-L is missing about 30 bp fromthe major CAP site, see below and Formula V and FIG. 7.

The immediate upstream sequence of the first ATG codon, GGACCATGC, fitswell with Kozak's consensus sequence GCC_(G) ^(A) CCATGG, for thetranslation initation site.

An in-frame ATG sequence was not found in the upstream sequence from theabove mentioned ATG sequence confirming that it is indeed the initiationcodon for the IRF-1 mRNA.

As mentioned above, no difference in nucleotide sequence was detectedbetween the cDNAs of λL28-8 and pIRF-L within the overlapping regions,except one nucleotide in the 3'-non-coding region.

The murine IRF-1 was thus found to consist of 329 amino acids with acalculated M.W. of 37.3 KD. Canonical N-glycosylation sites do notappear within the sequence.

No significant homology to other known proteins was detected bysearching in Protein Sequence Database (Natl. Biomed. Res. Found.,Washington, D.C.) and more recently published sequences.

Hydropathy plot analysis according to Kyte and Doolittle, 1982,indicates that the protein as a whole is highly hydrophilic (FIG. 4B).

Inspection of the deduced primary sequence of the murine IRF-1 revealsthe following features:

The amino terminal half, extending to amino acid (a.a.) 140 is rich inlysin (Lys) and arginine (Arg). In fact 31 out of 39 of the total Lysand Arg residues are located in this region. In the lower panel of FIG.4B is represented a diagrammatic summary of the location of the basicamino acids (Arg, Lys) (upward columns) and acidic amino acids (Asp,Glu) (downward columns).

As shown in FIG. 4B, this region shows strong hydrophilicity and isconsidered to be the region primarily responsible for the binding ofIRF-1 to the specific DNA sequences.

In this connection, characteristic motifs for many DNA binding proteinssuch as Zinc fingers and helix-turn-helix motifs (Pabo and Sauer, 1984,Evans and Hollenberg, 1988) were not detectable in the IRF-1 protein.

In contrast the rest of the molecule (i.e. the carboxyl terminal half)shows a relative abundance of aspartic acid (Asp), glutamic acid (Glu),Serine (Ser) and Threonine (Thr). Of 189 amino acids (from a.a. 140 to329), 33 (17%) represent acidic amino acids and 36 (19%) represent Serand Thr. Notably at cluster of 5 consecutive acidic amino acids is foundin a.a. 227 to 231. With regard to Ser and Thr, many appear to formclusters (region at a.a. 153-156, 190-192, 206-208, 220-222; referred toas the S-T regions herein). The S-T regions are depicted by small openrectangles in the lower panel of FIG. 4B.

Structure of the cDNA encoding human IRF-1

Following a procedure similar to that described above for the murineIRF-1, human IRF-1 cDNA was cloned and sequenced.

A human CDNA library was prepared by synthesising cDNA using poly(A)⁺RNA from a human T cell line Jurkat. The double stranded CDNA synthesisand subsequent cloning into plasmid vector CDM8 was carried outaccording to the procedure of Aruffo and Seed (Proc. Natl., Acad. Sci.,U.S.A., Vol. 84, pp 8573-8577, 1987) and Seed (Nature, Vol. 329, pp840-842, 1987).

The recombinant plasmids were introduced into E.coli strain, MC1061/p3using the procedure of Aruffo and Seed as mentioned above.

Clones of cDNA that cross-hybridize with mouse IRF-I cDNA were screenedusing λL28-8 cDNA (³² p-labeled) as the probe under low stringentconditions for DNA-DNA hybridization. The conditions employed wereexactly as described by Kashima et al. (Nature, Vol. 313, pp 402-404,1985). Hybridization was performed at 65° C. for 20 hours in a mediumcontaining 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodiumdodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovineserum albumin, and 50 μg/ml E. coil DNA as described in Ohno, S. et al.,Proc. Natl. Acad. Sci. U.S.A. 78:5305-5309 (1980) except that thewashing of the filter after hybridization was carried out in 3×SSC at65° C.

From the positive clones clone pHIRF31 containing the longest cDNAinsert was selected.

The desired cDNA sequence of clone pHIRF31 was isolated by digestion ofthe plasmid DNA by XhoI and after isolation subjected to sequencing bythe methods described above. The structure of the human IRF-1 gene isshown in Formula II and in FIG. 8.

The sequences for the deduced murine and human IRF-1 are shownjuxtaposed for comparison in FIG. 5.

Analysis of the human DNA revealed that this IRF-1 is shorter than themurine IRF-1 by four amino acids.

Strong conservation of the amino acid sequences can be seen between thetwo IRF-1 molecules. In particular, 133 out of 140 amino acids (95%) ofthe amino terminal halves can be seen to be identical.

Taken together, the above observation indicate that IRF-1 is a new classof DNA binding protein.

It should also be noted that the sequence ATTTATTTA and ATTTA, found inmany cytokine and proto-oncogene mRNAs, are present within the 3'non-translated region of the murine IRF-1 CDNA, and likewise thesequence ATTTA is found within the corresponding region of the humanIRF-1 cDNA. These sequences are believed to play a role in thepost-transcriptional regulation of gene expression by conferinginstability to the mRNA (Shaw and Kamen, 1986; Caput et al., 1986).

Plasmid pIRF-L was transfected into E.coli MC1061/p3 which was depositedas E.coli MC106/p3 (pIRF-L) at the Fermentation Research InstituteAgency of Industrial Science and Technology (FRI), 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken 305, Japan, under the terms of the BudapestTreaty on 19th Aug. 1988 under No. FERM BP-2005.

Plasmid pIRF31 was similarly transfected into E.coli MC1061/p3 which wasdeposited as E.coli MC106/p3 (pIRP-31) at the FRI under the BudapestTreaty on 19th Aug. 1988 under No. FERM BP-2006.

Regulation of the IRF gene

1. Expression of IRF-1 mRNA

In view of the fact that IRF-1 manifests affinities to regulatorysequences of genes other than IFN-β gene and is thus involved in theregulation of a set of genes in various cell types, examination of theexpression of the IRF-1 mRNA in mouse cells derived from various tissuesand organs was carried out using the murine cDNA as a probe. To preparethis probe M13 mp10 phage DNA (see below) containing the sense strand ofIRF-1 gene PstI fragment was used as a template to synthesise the ³²p-labeled antisense DNA, the product was digested by EcoRI and the probeDNA isolated as described by Fujita et al. (1985).

Total RNA was isolated by the established procedure of Aruffo and Seed1987.

The blotting analysis was then carried out essentially as described byThomas (1980), the x-ray film being exposed for 3 days and the resultsare shown in FIG. 6A. The various lanes represent the results of runscarried out using whole cell RNA from the following tissue:

    ______________________________________                                        Lane      1          Brain                                                    Lane      2          Heart                                                    Lane      3          Liver                                                    Lane      4          Lung                                                     Lane      5          Spleen (unstimulated)                                    Lane      6          Thymus                                                   Lane      7          Kidney                                                   Lane      8          Muscle                                                   Lane      9          Intestine                                                Lane      10         Spleen (unstimulated)                                    Lane      11         ConA - stimulated spleen                                 ______________________________________                                    

In the run for each lane 5 μg of whole cell RNA was used, except in lane8 for which only 1.2 μg RNA was used.

It will be seen from FIG. 6A that a band corresponding to about 2.0 kbwas detected in most of the RNA samples by this blotting analysis,although the mRNA expression level seems low. It is noteworthy that themRNA expression level in the spleen-derived lymphocytes was augmenteddramatically following stimulation by ConA (Lane 11).

In a further assay mouse L929 cells were induced by NDV as describedpreviously (Fujita et al., 1985) and the cytoplasmic RNA extracted, bythe procedure of Aruffo and Seed 1987, every three hours afterinfection.

Probe DNAs were prepared from the following various sequences andlabeled by the multiprime labeling reaction (Amersham), namely

(i) an 1.8 kb EcoRI fragment from λL28-8 (specific activity 2×10⁸cpm/μg);

(ii) a 0.5 kb BamHI-BglII fragment from a mouse IFN-β genomic clone(specific activity 5×10⁸ cpm/μg) and

(iii) a 2.0 kb BamHI-PvuII fragment of a clone containing human β-actinpseudogene (specific activity 5×10⁸ cpm/μg).

The results are shown in FIG. 6B. Blotting analysis was carried out, asdescribed above, using the procedure of Thomas (1980).

Each lane received 10 μg of the cytoplasmic RNA. The x-ray film wasexposed for 3 hours. Densitometric analysis revealed that IRF-1 mRNAincreased about 25-fold, 9-12 hours after NDV infection.

Whilst the increase in mRNA is dramatic it is transient, peaking at 9 to12 hours and levelling off 15 hours after induction. mRNA accumulationpreceeds the accumulation of the IFN-β mRNA; as can be seen from FIG. 6Bthe induction of the IRF-1 mRNA can be observed already 3 hours afterNDV infection, while the IFN-β mRNA is detectable only after 6 hoursunder similar blotting conditions for both RNAs.

The IRF-1 promoter

As demonstrated above, the IRF-1 gene is transcriptionally regulated byvarious agents such as viruses and mitogenes.

Southern blot analysis of the chromosomal DNA indicated that the IRF-1gene may be spliced and not multimembered in the mouse.

A λphage library containing new-born mouse DNA was screened for theclones harboring the IRF-1 promoter sequence using the same λL28-8derived cDNA probe used above. Four positive clones were identified, allof which were found to contain the same genomic DNA and one of themλg14-2 was used for further analysis. A PstI fragment was sub-clonedinto the PstI site of pUC19 to construct p19IRFP.

The same DNA was thereafter cloned into the PstI site of M13mp10 andM13mp11 which were used to generate DNA for sequence analysis.

Nucleotide sequence analysis of the PstI fragment from the above cloneswas carried out as previously described. Major and minor CAP sites wereidentified by S1 mapping analysis.

The determined sequence is shown in FIG. 7A. As can be seen thedownstream sequence of the DNA perfectly matches that of the pIRF-Lderived cDNA.

The S1 nuclease analysis indicates the presence of two CAP sites for theIRF-1 mRNA in which the major site is about 20 nucleotides downstream ofthe minor site. Typical TATA box sequences are not present within theupstream region of the gene. In view of the unusual abundancy of CpGsequence, this region probably constitutes an "HTF island" (Bird, 1986).

The promoter region contains two GC boxes and one CAAT box (see FIG.7A); the former boxes should bind SpI (Kadogan et al., 1986) and thelatter, CP-1 or CP-2 (Chodosh et al., 1988).

The PstI fragment containing the promoter sequences was then tested forits reactivity in response to extracellular signals e.g. virusinducibility in the following manner:

A chimeric gene was constructed in which a reporter gene, namely abacterial chloramphenicol acetyltransferase (CAT) gene was abutteddownstream of the PstI segment. This was done by excising a PstIfragment from P19IRFP (see above) by BamHI and HindIII and cloning theresulting fragment into the BglII-HindIII backbone fragment of pA₁₀ cat₂(Rosenthal et al., 1983) to construct pIRFcat.

Several further constructs were prepared as follows:

pIRFΔcat was prepared by digesting the p19IRFP-derived BamHI-HindIIIfragment with HaeIII whose single recognition site is located at -30 to-35 from the major CAP site (FIG. 7A). The resulting HaeIII-HindIIIfragment was ligated with the BglII-HindIII backbone fragment of pA₁₀cat₂ and the following synthetic DNA ##STR11##

Thus both pIRFcat and pIRFΔcat contained sequences up to -320 and -48from the major CAP site respectively.

p-125cat contains the promoter sequence of the human IFN-β gene asdescribed by Fujita et al., 1987.

pSV2cat is described in Gorman et al., Science, Vol. 221, pp 551-553.

As a reference gene PRSVgpt was used (see Gorman et al., above).

The various genes were transfected into mouse L929 cells using thecalcium phosphate method (Fujita et al., 1985). 5×10⁶ cells weretransfected with 7.5 μg of the test plasmid containing the CAT reportergene and 2.5 μg of pRSVgpt. The cells were induced by NDV ormock-induced and then subjected to the enzyme assay as described byFujita et al., 1985.

In calculating the relative CAT activity, CAT activity from themock-induced cells, transfected with pSV2cat was taken as 100%. Each CATactivity was normalised by the Ecogpt (Mulligan and Berg, Proc. Natl.Acad. Sci. U.S.A., Vol. 78, pp 2072-2076) of the respective samples. Insamples where the CAT was below the background level, they were markedas b.b.

The results are shown in FIG. 7B.

It will be seen that transfection of the pIRFcat into mouse L929 gaverise to the expression of low level CAT activity. The CAT expressionlevel was increased when the transfected cells were stimulated by NDV.Deletion of the 300 bp upstream sequence of the IRF-1 gene (pIRFΔcat)virtually abolished both constitutive and induced expression of the CATgene. This demonstrates that the promoter sequence lies within the 300bp upstream region and is virus inducible.

Construction of expression plasmids

1. Phage DNA of clone λL28-8 was digested by EcoRI and the cDNA insertwas recovered. The EcoRI sites of the cDNA were rendered flush by T4 DNApolymerase and then ligated with synthetic adaptor DNAs having thesequence pGATCCATTGTGCTGG and pCCAGCACAATG according to Aruffo and Seed,1987.

After removal of the synthetic DNAs by 5-20% potassium acetate gradientcentrifugation (Aruffo and Seed, 1987), the IRF-1 cDNA with the adaptorDNAs attached to both its ends was ligated with BstXI-cleaved CDM8vector DNA (Seed, B., Nature, vol. 329, pp 840-842, 1987). PlasmidspIRF-S and pIRF-A containing the IRF-1 cDNA in the sense and antisenseorientation with respect to the CMV promoter respectively were isolated.

Each plasmid DNA was co-transfected with either p-55cat or p55ClB(Fujita et al., 1987) into L929 cells and the CAT expression level wasdetermined.

The results are shown in Table 1 below:

                  TABLE 1                                                         ______________________________________                                                Transfected                                                                           Induction   CAT activity                                              plasmids                                                                              by NDV      (% conversion)                                    ______________________________________                                                  pIRF-S    -           <1%                                                     p-55cat                                                                       pIRF-S    -           50 %                                                    p-55C1B                                                             Exp. 1    pIRF-A    -           <1%                                                     p-55cat                                                                       pIRF-A    -           <1%                                                     pIRF-S    -           1,7%                                                    p-55C1B                                                                       pIRF-S    +           3,6%                                                    p-55C1B                                                             Exp. 2    pIRF-A    -           <0,1%                                                   p-55C1B                                                                       pIRF-A    +           <0,1 %                                                  p-55C1B                                                             ______________________________________                                    

The DNA transfection efficiency varies depending on the state of therecipient cells (in this case, mouse L929 cells). The efficiency wasmuch lower in Exp. 2 compared to Exp. 1. Therefore, the CAT expressionlevel is relatively lower in Exp. 2.

As can be seen from the above table significant CAT activity wasdetectable only in the cells transfected by p-55CIB and pIRF-S. Theydemonstrate that the IRF-1 binds to the repeated (8 times) AAGTGAsequences present in the upstream of the CAT gene in p-55CIB and therebypromotes transcription of the distal CAT gene.

The results further show that the CAT expression level is increased(more than two fold) by infecting the transfected cells with NDV (Table1), demonstrating that it is possible to control the gene expression byvarious stimuli such as viruses.

An expression plasmid for the production of a protein consisting ofIRF-1 DNA binding domain and transcriptional activation domain of yeastGAL4 was constructed as follows:

Plasmid PIRF-S was digested by HindIII and PstI and the cDNA insertisolated. The cDNA was digested by DraIII and the HindIII-DraIIIfragment (about 550 bp) was recovered and designated Fragment A.

The expression vector CDM8 was digested by HindIII and XbaI and thebackbone DNA isolated and designated Fragment B.

The DNA encoding the yeast GAL4 transcriptional activation domain wasisolated from plasmid pRB968 (Ma and Ptashne, 1987) as follows:

The pRB968 DNA was first digested by HindIII and the termini wererendered flush by T4 DNA polymerase. Synthetic XbaI linker DNA was addedto the DNA and the DNA was subsequently digested by PvuII and XbaI.

The resulting ca. 600 bp PvuII-XbaI DNA fragment was recovered anddesignated Fragment C.

In addition, a synthetic DNA with the following sequence was prepared:##STR12## designated Fragment D.

An expression vector pIRFGAL4 was constructed by ligating the FragmentsA, B, C and D. As a control plasmid, plasmid PIRFΔGAL4 was constructedby ligating Fragments A, B, C and a synthetic DNA with the followingsequence: ##STR13##

As a terminator triplet, TGA, is present in frame between the IRF-1 andGAL4 sequences in pIRFΔGAL4, the expressed protein should lack the GAL4activation domain.

In order to test the functional properties of the plasmid encodedchimeric transcriptional factor pIRFGAL4 and pIRFΔGAL4 were eachco-transfected with p-55CIB into L929 cells and CAT expressionmonitored. The results are shown in Table 2 below:

                  TABLE 2                                                         ______________________________________                                        Transfected plasmid                                                                         CAT activity (% conversion)                                     ______________________________________                                        pIRF-s        2,0%                                                            p-55C1B                                                                       pIRF-A        <0,2%                                                           p-55C1B                                                                       pIRFGAL       1,4%                                                            p-55C1B                                                                       pIRF .increment. GAL4                                                                       <0,2%                                                           p-55C1B                                                                       ______________________________________                                    

Host cell, Mouse L929 cells. Cells were not induced by NDV.

The results show that the expression of a target gene (such as a genesencoding an interleukin, an interferon (α, β and γ), a plasminogenactivator, erythropoietin, granulocyte colony stimulating factor,insulin, human growth hormone or superoxide dismutase (or mutants of thehuman genes) can be augmented by IRF-1.

Target genes as mentioned above, such as interferon genes e.g. IFN-α,IFN-β, IFN-γ, IFN-omega and plasminogen activators e.g. t-PA,pro-urokinase or urokinase etc. can be expressed more efficiently alsoby including therefor promoters fused with various lengths ofrecognition sequences for IRF-1, e.g. AAGTGA.

For example the target genes can be introduced into various host cells,together with either intact IRF-1 or chimeric IRF-1 genes. By increasingthe length of IRF-1 recognition site DNA e.g. by increasing the numberof AAGTGA repeats and the expression level of the transcription factor ahigh-level expression of the target genes can be achieved.

For example the AAGTGA repeat sequences can be abutted to a suitablepromoter such as IFN-β promoter or SV40 virus early promoter. A targetgene e.g. a t-PA gene or IFN-β gene can be linked downstream of such apromoter; the structure of such a constructed gene would be

    (AAGTGA).sub.x (Promoter) (target gene e.g. t-PA gene).

Such a gene could then be introduced into and amplified in CHO cellse.g. CHO DXBII (dhfr-strain) cells (Urlaub and Chasin, Proc. Natl.,Acad. Sci., U.S.A., Vol. 77, pp 4216-4220, 1980).

Ideally, as discussed above, a host cell will be chosen from a cellwhich has no or substantially no level of endogenous IRF-1 activity. TheIRF-1 gene, preferably either with a strong promoter such as CMVpromoter or an inducible promoter such as metallothionen gene promotercan be introduced into the various host cells in a conventional manner.

The IRF-1 gene can be co-introduced and amplified together with thetarget gene. Alternatively the IRF-1 gene and the target gene can beseparately introduced into the host cell.

In such transfected cells, IRF-1 may be produced either constitutively(in the case of e.g. CMV promoter) or in an induced manner (in the caseof e.g. the metallothionen promoter it is induced by divalent metalssuch as zinc). The expressed IRF-1 binds to the AAGTGA repeats andaugments the distal target gene e.g. t-PA gene or IFN gene.

Such expression could be further augmented by virus e.g. NDV induction,as can be seen from Table 1, Experiment 2 such induction increases theactivity of the IRF-1.

Mouse cDNA sequence which cross-hybridises with murine IRF-1 cDNA ofFormula III

A mouse cDNA library was prepared by the procedure described above. ThecDNA was synthesised by the standard procedure using the mouse L929cell-derived mRNA and the CDNA was inserted into the λgt11 vector by thestandard procedure. The resulting λgt11 library was then screened toisolate cDNA clones whose inserts cross-hybridize with the murine IRF-1cDNA described above, as follows:

Nitrocellulose filters containing the phage plaque DNAs were incubatedin the following stages

(1) in 3×SCC at 65° C. for 30 minutes, followed by

(2) 60 minutes incubation in 3×SSC containing Denhart's solution (0.2%bovine serum albumin, 0.2% Ficoll, 0.2% polyvinylpyrrolidone 25),followed by

(3) a pre-hybridisation step consisting of incubation for 12 hours at65° C. in a solution containing 1M NaCl, 50 mM Tris-HCl, pH 8.0, 10 mMEDTA, 0.1% SDS, 50 μg/ml single stranded carrier DNA (e.g. salmon spermDNA) and Denhart's solution and

(4) the stage 3 incubation was repeated but including ³² p-labeledmurine IRF-1 cDNA as a probe. This cDNA probe was prepared as the EcoRIcleaved insert from λL-28-8 and translated by the Multiprime labelingsystem (see above). The incubation was carried out at 65° C. for 12hours.

The filters were then washed, rinsed briefly in 2×SCC solution and thenwashed in 3×SCC solution containing 0.1% SDS for 30 minutes at 65° C.This procedure was twice repeated.

One of the positive clones, designated pHH-45 was selected and wasrevealed to contain cDNA covering only part of a coding sequence for anIRF.

The cDNA insert in PHH-45 was therefore isolated and used to screenclones containing larger inserts as described above for the preparationof pIRF-L under the heading "Structure of the cDNA encoding murineIRF-1".

Of the positive clones identified the one designated pIRF2-5 wasselected and characterised using the procedure previously described formurine IRF-1. The complete hybridizing cDNA sequence is shown in formulaIVa and the amino acid sequence of the corresponding IRF protein isshown in formula VIII.

Plasmid pIRF 2-5 was transfected into E.coli MC 1061/p3 which wasdeposited as E.coli at the FRI under the Budapest Treaty on 22 Nov. 1988under No. FERM BP-2157.

cDNA from the yeast genome which hybridizes with human IRF-1 cDNAsequence

Yeast DNA was prepared by the standard procedure and digested withEcoRI. 5 μg of the digested DNA was loaded onto 0.8% agarose gel andsubjected to electrophoresis and DNA blotting by standard procedures.

The blotted filter was treated exactly as described in the precedingexample for isolating mouse DNA which hybridizes with murine IRF-1,except as follows:

In step (3) the incubation temperature was 55° C. and in step (4) theincubation was also carried out at 55° C. and the radioactive probe wasthe human IRF-1 cDNA isolated from pHIRF31 by XhoI digestion of theplasmid, this probe being labeled as described in the preceding examplefor murine IRF-1. The filter was washed at 55° C. in 2×SSC. The positiveclones were identified by autoradiography (see FIG. 9).

REFERENCES

Abreu S. L., Bancroft F. C., and Stewart II E. W. (1979). Interferonpriming. J. Biol. Chem. 254, 414-418.

Aruffo A., and Seed B. (1987). Molecular cloning of a cDNA by ahigh-efficiency COS cell expression system. Proc. Natl. Acad. Sci.U.S.A. 84, 8573-8577.

Bird A. P. (1986). CpG-rich islands and the function of DNA methylation.Nature 321, 209-213.

Caput D., Beutler B., Hartog K., Thayer R., Brown-Schimer S., and CeramiA. (1986). Identification of a common nucleotide sequence in the 3'untranslated region of mRNA molecules specifying inflammatory mediators.Proc. Natl. Acad. Sci U.S.A. 83, 1670-1674.

Cavalieri R. L., Havell E. Z., Vilcek J., and Pestka S. (1977).Induction and decay of human fibroblast interferon mRNA. Proc. Natl.Acad. Sci. U.S.A. 74, 4415-4419.

Chodosh L. A., Baldwin A. S., Carthew R. W., and Sharp P. A. (1988).Human CCAAT-binding proteins have heterologous subunits. Cell 53, 11-24.

Dinter H., Hauser H., Mayr U., Lammers R., Bruns W., Gross G., andCollins J. (1983). Human interferon-beta and co-induced genes: molecularstudies. In the biology of the Interferon System 1983, E. De Maeyer andH. Schellekens, eds. (Amsterdam: Elsevier Science Publishers), 33-34.

Dinter H., and Hauser H. (1987). Cooperative interaction of multiple DNAelements in the human interferon-β promoter. Eur. J. Biochem. 166,103-109.

Evans R. M., and Hollenberg S. M. (1988). Zinc Fingers: Gilt byassociation. Cell 52, 1-3.

Fujita T., Saito S., and Kohno S. (1979). Priming increases the amountof interferon mRNA in poly(rI):poly(rC)-treated L cells. J. Gen. Viol.45,301-308.

Fujita T., Ohno S., Yasumitsu H., and Taniguchi T. (1985). Deliminationand properties of DNA sequences required for the regulated expression ofhuman interferon-β gene Cell 41, 489-496.

Fujita T., Shibuya H., Hotta H., Yamanishi K., and Taniguchi T. (1987).Interfero-β gene regulation: Tandemly repeated sequences of a synthetic6 bp oligomer function as a virus-inducible enhancer. Cell 49, 357-367.

Galabru J., and Hovanessian A. G. (1985). Two interferon-β indecedproteins are involved in the protein kinase complex dependent ondouble-stranded RNA. Cell 43, 685-694.

Goodbourn S., Zinn K., and Maniatis T. (1985). Human β-interferon geneexpression is regulated by an inducible enhancer element. Cell 41,509-520.

Huynh T. V., Young R. A., and Davis R. W. (1985). Constructing andscreening cDNA libraries in gt10 and 11. In DNA cloning-A PracticalApproach, Volume 1, D. M. Glover, ed. (Oxford: IRL Press), pp. 49-78.

Israel A., Kimura A., Fournier A., Fellous M., and Kourilsky P. (1986).Interferon response sequence potentiates activity of an enhancer in thepromoter region of a mouse H-2 gene. Nature 322, 743-746.

Kadonaga J. T., Jones K. A., and Tjian R. (1986). Promoter-specificactivation of RNA polymerase II transcription by Sp 1. Trends Biochem.Sci. 11, 20-23.

Kakidani H., and Ptashne M. (1986). GAL4 activates gene expression inmammalian cells. Cell 52, 161-167.

Keller A. D., and Maniatis T. (1988). Identification of an induciblefactor that binds to a positive regulatory element of the humanβ-interferon gene. Proc. Natl. Acad. Sci U.S.A. 85, 3309-3313.

Kohase M., May L. T., Tamm I., Vilcek J., and Sehgal P. B. (1987). Acytokine network in human diploid fibroblasts: interactions of betainterferons, tumor necrosis factor, platelet-derived growth factor andinterleukin-1. Mol. Cell. Biol. 7, 272-280.

Korber B., Mermod N., Hood L., and Stroynowski I. (1988). Regulation ofgene expression by interferons: Control of H-2 Promoter responses.Science 239, 1302-1306.

Kozak M. (1987). An analysis of 5'-noncoding sequences from 699vertebrate messenger RNAs. Nucl. Acids Res. 15, 8125-8143.

Krebs E., Eisenman R., Kuenzel E., Litchfield D., Lozeman F., LiischerB., and Sommercorn J. (1988). Casein Kinase II as a potentiallyimportant enzyme concerned with signal transduction. In the MolecularBiology of Signal Transduction (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory) Abstract p.35.

Kuhl D., de la Fuente J., Chaturvedi M., Parimoo S., Ryals J., Mayer F.,and Weissmann C. (1987). Reversible silencing of enhancers by sequencesderived from the human IFN-α promoter. Cell 50, 1057-1069.

Kyte J., and Doolittle R. F. (1982). A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157, 105-132.

Levy D. E., Kessler D. S., Pine R., Reich N., and Darnell J. E. (1988).Interferon-induced nuclear factors that bind a shared promoter elementcorrelate with positive and negative transcriptional control. Genes andDevelopment 2, 383-393.

Ma J., and Ptashne M. (1987). The carboxy-terminal 30 amino acids ofGAL4 are recognised by GAL80. Cell 50, 137-142.

Maxam A., and Gilbert W. (1980). Sequencing end-labeled DNA with basespecific chemical cleavages. Meth. Enzym. 65, 499-560.

Moore R. N., Larsen H. S., Horohov D. W., and Rouse B. T. (1984).Endogenous regulation of macrophase proliferative expansion bycolony-stimulation-factor-induced interferon. Science 223, 178-180.

Nedwin G., Naylor S., Sakaguchi A., Smith D., Jarrett-Nedsin J., PennicaD., Goeddel D., and Gray P. (1985). Human lymphotoxin and tumor necrosisfactor genes: structure, homology and chromosomal localisation. Nucl.Acids Res. 13, 6361-6373.

Nir U., Cohen B., Chen L., and Revel M. (1984). A human IFN-β1 genedeleted of promoter sequences upstream from the TATA box is controlledpost-transcriptionally by dsRNA. Nucl. Acids Res. 12, 6979-6993.

Ohno S., and Taniguchi T. (1983). The 5'-flanking sequence of humaninterferon-β gene is responsible for viral induction of transcription.Nucl. Acids Res. 11, 5403-5412.

Onozaki K., Urawa H., Tamatani T., Iwamura Y., Hashimoto T., Baba T.,Suzuki H., Yamada M., Yamamoto S., Oppenheim J. J., and Matsushima K.(1988).Synergistic interactions of interleukin 1, interferon-β and tumornecrosis factor in terminally differentiating a mouse myeloid leukemiccell line (M1). J. Immunol. 140, 112-119.

Pabo C. O., and Sauer R. T. (1984). Protein-DNA recognition. Ann. Rev.Biochem. 53, 293-321.

Raji N. B. K., and Pitha P. M. (1981). An analysis of interferon mRNA inhuman fibroblast cells induced to produce interferon. Proc. Natl. Acad.Sci U.S.A. 78, 7426-7430.

Raji N. B. K., and Pitha P. M. (1983). Two levels of regulation ofβ-interferon gene expression in human cells. Proc. Natl. Acad. SciU.S.A. 80, 3923-3927.

Resnitzky D., Yarden A., Zipori D., and Kimchi A. (1986). Autocrineβ-related interferon controls c-myc suppression and growth arrest duringhematopoietic cell differentiation. Cell 46, 31-40.

Rosenthal N., Kress M., Gruss P., and Khoury G. (1983). BK viralenhancer element and a human cellular homolog. Science 222, 749-755.

Ryals J., Dieks P., Ragg H., and Weissmann C. (1985). A 46-nucleotidepromoter segment from an INF-α gene renders an unrelated promoterinducible by virus. Cell 41, 497-507.

Shaw G., and Kamen R. (1986). A conserved AU sequence from the 3'untranslated region of GM-CSF mRNA mediates selective mRNA degradation.Cell 46, 659-667.

Singh H., LeBowitz J. H., Baldwin Jr. A. S., and Sharp P. A. (1988).Molecular cloning of an enhancer binding protein: Isolation by screeningof an expression library with a recognition site DNA. Cell 52 415-423.

Sugita K., Miyazaki J. I., Appella E., and Ozato K. (1987). Interferonsincrease transcription of a major histocompatibility class I gene via a5' interferon consensus sequence. Mol. Cell. Biol. 7, 2625-2630.

Taniguchi T., Matsui H., Fujita T., Takaoka C., Kashima N., YoshimotoR., and Hamuro J. (1983). Structure and expression of a cloned cDNA forhuman interleukin-2. Nature 302, 305-310.

Taniguchi T. (1988). regulation of cytokine gene expression. Ann. Rev.Immunol. 6, 439-464.

Thomas P. S. (1980). Hybridisation of denatured RNA and small DNAfragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A.77, 5201-5205.

Tiwari R. J., Kusari J., and Sen G. C. (1987). Functional equivalents ofinterferon-mediated signals needed for induction of a mRNA can begenerated by double-stranded RNA and growth factors. EMBO J. 6,3373-3378.

Warren M. K., and Ralf P. (1986). Macrophage growth factor CSF-1stimulates human monocyte production of interferon, tumor necrosisfactor and colony stimulating activity. J. Immunol. 137, 2281-2285.

Webster N., Jin J. R., Green S., Hollis M., and Chambon P. (1988). Theyeast UAGs is a transcriptional enhancer in human HeLa cells in thepresence of the GAL4 trans-activator. Cell 52, 169-178.

Weissmann C., and Weber H. (1986). The interferon genes. Proc. Natl.Acid Res. Mol. Biol., 33, 251-300.

Young R. A., and Davis R. W. (1983). Yeast RNA polymerase II genes:Isolation with antibody probes. Science 222, 778-782.

Zinn K., Dimaio D., and Maniatis T. (1983). Identification of twodistinct regulatory regions adjacent to the human β-interferon gene.Cell 34, 865-879.

Zullo J. N., Cochan B. H., Huang A. S., and Stiles C. D. (1985).Platelet-derived growth factor and double-stranded ribonucleic acids andstimulate expression of the same genes in 3T3 cells. Cell 43, 793-800.

We claim:
 1. A composition comprising proteins extracted from arecombinant host cell, wherein said composition comprises a recombinantIRF-1 protein produced by said cell and wherein said IRF-1 can bind to(a) a first recognition sequence (AAGTGA)₄ and (b) a second recognitionsequence that is at bases -64 to -100 of the human IFN-β gene, whereinsaid binding in step (a) and (b) augments transcription of a codingsequence operably linked to a promoter that contains said first orsecond recognition sequence, and wherein said IRF-1 protein is encodedby a recombinant nucleic acid sequence in said host cell that hybridizesto the anti sense sequence of a DNA selected from the group consistingof ##STR14## ##STR15## when the hybridization is performed at 65 degreesCentigrade for 20 hours in a medium consisting essentially of 1M NaCl,50 ml Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate, 0.2%ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 50 μg/mlE. coli DNA, said nucleic acid sequence and said antisense sequence. 2.The composition of claim 1, wherein the amino acid sequence of saidIRF-1 is ##STR16##
 3. The composition of claim 2, wherein said nucleicacid sequence comprises ##STR17##
 4. The composition of claim 3, whereinsaid nucleic acid sequence comprises upstream and downstream sequencesthat flank the sequence encoding said IRF-1, and said upstream anddownstream sequences and said IRF-1 encoding sequences comprise##STR18##
 5. The composition of claim 1, wherein the amino acid sequenceof said IRF-1 comprises

    __________________________________________________________________________    Met                                                                              Pro                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Met                                                                              Arg                                                                              Met                                                                              Arg                                                                              Pro                                                                              Trp                                                                              Leu                                                                              Glu                                                                              Met                                                                              Gln                                                                              Ile                              Asn                                                                              Ser                                                                              Asn                                                                              Gln                                                                              Ile                                                                              Pro                                                                              Gly                                                                              Leu                                                                              Ile                                                                              Trp                                                                              Ile                                                                              Asn                                                                              Lys                                                                              Glu                                                                              Glu                                                                              Met                              Ile                                                                              Phe                                                                              Gln                                                                              Ile                                                                              Pro                                                                              Trp                                                                              Lys                                                                              His                                                                              Ala                                                                              Ala                                                                              Lys                                                                              His                                                                              Gly                                                                              Trp                                                                              Asp                                                                              Ile                              Asn                                                                              Lys                                                                              Asp                                                                              Ala                                                                              Cys                                                                              Leu                                                                              Phe                                                                              Arg                                                                              Ser                                                                              Trp                                                                              Ala                                                                              Ile                                                                              His                                                                              Thr                                                                              Gly                                                                              Arg                              Tyr                                                                              Lys                                                                              Ala                                                                              Gly                                                                              Glu                                                                              Lys                                                                              Glu                                                                              Pro                                                                              Asp                                                                              Pro                                                                              Lys                                                                              Thr                                                                              Trp                                                                              Lys                                                                              Ala                                                                              Asn                              Phe                                                                              Arg                                                                              Cys                                                                              Ala                                                                              Met                                                                              Asn                                                                              Ser                                                                              Leu                                                                              Pro                                                                              Asp                                                                              Ale                                                                              Glu                                                                              Glu                                                                              Val                                                                              Lys                                                                              Asp                              Gln                                                                              Ser                                                                              Arg                                                                              Asn                                                                              Lys                                                                              Gly                                                                              Ser                                                                              Ser                                                                              Ala                                                                              Val                                                                              Arg                                                                              Val                                                                              Tyr                                                                              Arg                                                                              Met                                                                              Leu                              Pro                                                                              Pro                                                                              Leu                                                                              Thr                                                                              Arg                                                                              Asn                                                                              Gln                                                                              Arg                                                                              Lys                                                                              Glu                                                                              Arg                                                                              Lys                                                                              Ser                                                                              Lys                                                                              Ser                                                                              Ser                              Arg                                                                              Asp                                                                              Thr                                                                              Lys                                                                              Ser                                                                              Lys                                                                              Thr                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Leu                                                                              Cys                                                                              Gly                                                                              Asp                                                                              Val                                                                              Ser                              Pro                                                                              Asp                                                                              Thr                                                                              Phe                                                                              Ser                                                                              Asp                                                                              Gly                                                                              Leu                                                                              Ser                                                                              Ser                                                                              Ser                                                                              Thr                                                                              Leu                                                                              Pro                                                                              Asp                                                                              Asp                              His                                                                              Ser                                                                              Ser                                                                              Tyr                                                                              Thr                                                                              Thr                                                                              Gln                                                                              Gly                                                                              Tyr                                                                              Leu                                                                              Gly                                                                              Gln                                                                              Asp                                                                              Leu                                                                              Asp                                                                              Met                              Glu                                                                              Arg                                                                              Asp                                                                              Ile                                                                              Thr                                                                              Pro                                                                              Ala                                                                              Leu                                                                              Ser                                                                              Pro                                                                              Cys                                                                              Val                                                                              Val                                                                              Ser                                                                              Ser                                                                              Ser                              Leu                                                                              Ser                                                                              Glu                                                                              Trp                                                                              His                                                                              Met                                                                              Gln                                                                              Met                                                                              Asp                                                                              Ile                                                                              Ile                                                                              Pro                                                                              Asp                                                                              Ser                                                                              Thr                                                                              Thr                              Asp                                                                              Leu                                                                              Tyr                                                                              Asn                                                                              Leu                                                                              GlN                                                                              Val                                                                              Ser                                                                              Pro                                                                              Met                                                                              Pro                                                                              Ser                                                                              Thr                                                                              Ser                                                                              Glu                                                                              Ala                              Ala                                                                              Thr                                                                              Asp                                                                              Glu                                                                              Asp                                                                              Glu                                                                              Glu                                                                              Gly                                                                              Lys                                                                              Ile                                                                              Ala                                                                              Glu                                                                              Asp                                                                              Leu                                                                              Met                                                                              Lys                              Leu                                                                              Phe                                                                              Glu                                                                              Gln                                                                              Ser                                                                              Glu                                                                              Trp                                                                              Gln                                                                              Pro                                                                              Thr                                                                              His                                                                              Ile                                                                              Asp                                                                              Gly                                                                              Lys                                                                              Gly                              Tyr                                                                              Leu                                                                              Leu                                                                              Asn                                                                              Glu                                                                              Pro                                                                              Gly                                                                              Thr                                                                              GlN                                                                              Leu                                                                              Ser                                                                              Ser                                                                              Val                                                                              Tyr                                                                              Gly                                                                              Asp                              Phe                                                                              Ser                                                                              Cys                                                                              Lys                                                                              Glu                                                                              Glu                                                                              Pro                                                                              Glu                                                                              Ile                                                                              Asp                                                                              Ser                                                                              Pro                                                                              Arg                                                                              Gly                                                                              Asp                                                                              Ile                              Gly                                                                              Ile                                                                              Gly                                                                              Ile                                                                              Gln                                                                              His                                                                              Val                                                                              Phe                                                                              Thr                                                                              Glu                                                                              Met                                                                              Lys                                                                              Asn                                                                              Met                                                                              Asp                                                                              Ser                              Ile                                                                              Met                                                                              Trp                                                                              Met                                                                              Asp                                                                              Ser                                                                              Leu                                                                              Leu                                                                              Gly                                                                              Asn                                                                              Ser                                                                              Val                                                                              Arg                                                                              Leu                                                                              Pro                                                                              Pro                              Ser                                                                              Ile                                                                              Gln                                                                              Ala                                                                              Ile                                                                              Pro                                                                              Cys                                                                              Ala                                                                              Pro.                                                  __________________________________________________________________________


6. The composition of claim 5, wherein said nucleic acid sequencecomprises

    __________________________________________________________________________    ATG                                                                              CCA                                                                              ATC                                                                              ACT                                                                              CGA                                                                              ATG                                                                              CGG                                                                              ATG                                                                              AGA                                                                              CCC                                                                              TGG                                                                              CTA                                                                              GAG                                                                              ATG                                                                              CAG                                                                              ATT                              AAT                                                                              TCC                                                                              AAG                                                                              CAA                                                                              ATC                                                                              CCA                                                                              GGG                                                                              CTG                                                                              ATC                                                                              TGG                                                                              ATC                                                                              AAT                                                                              AAA                                                                              GAA                                                                              GAG                                                                              ATG                              ATC                                                                              TTC                                                                              CAG                                                                              ATT                                                                              CCA                                                                              TGG                                                                              AAG                                                                              CAC                                                                              GCT                                                                              GCT                                                                              AAG                                                                              CAC                                                                              GGC                                                                              TGG                                                                              GAC                                                                              ATC                              AAC                                                                              AAG                                                                              GAT                                                                              GCC                                                                              TGT                                                                              CTG                                                                              TTC                                                                              CGG                                                                              AGC                                                                              TGG                                                                              GCC                                                                              ATT                                                                              CAC                                                                              ACA                                                                              GGC                                                                              CGA                              TAC                                                                              AAA                                                                              GCA                                                                              GGA                                                                              GAA                                                                              AAA                                                                              GAG                                                                              CCA                                                                              GAT                                                                              CCC                                                                              AAG                                                                              ACA                                                                              TGG                                                                              AAG                                                                              GCA                                                                              AAC                              TTC                                                                              CGT                                                                              TGT                                                                              GCC                                                                              ATG                                                                              AAC                                                                              TCC                                                                              CTG                                                                              CCA                                                                              GAC                                                                              ATC                                                                              GAG                                                                              GAA                                                                              GTG                                                                              AAG                                                                              GAT                              CAG                                                                              AGT                                                                              AGG                                                                              AAC                                                                              AAG                                                                              GGC                                                                              AGC                                                                              TCT                                                                              GCT                                                                              GTG                                                                              CGG                                                                              GTG                                                                              TAC                                                                              CGG                                                                              ATG                                                                              CTG                              CCA                                                                              CCC                                                                              CTC                                                                              ACC                                                                              AGG                                                                              AAC                                                                              CAG                                                                              AGG                                                                              AAA                                                                              GAG                                                                              AGA                                                                              AAG                                                                              TCC                                                                              AAG                                                                              TCC                                                                              AGC                              CGA                                                                              GAC                                                                              ACT                                                                              AAG                                                                              AGC                                                                              AAA                                                                              ACC                                                                              AAG                                                                              AGG                                                                              AAG                                                                              CTG                                                                              TGT                                                                              GGA                                                                              GAT                                                                              GTT                                                                              AGC                              CCG                                                                              GAC                                                                              ACT                                                                              TTC                                                                              TCT                                                                              GAT                                                                              GGA                                                                              CTC                                                                              AGC                                                                              AGC                                                                              TCT                                                                              ACC                                                                              CTA                                                                              CCT                                                                              GAT                                                                              GAC                              CAC                                                                              AGC                                                                              AGT                                                                              TAC                                                                              ACC                                                                              ACT                                                                              CAG                                                                              GGC                                                                              TAC                                                                              CTG                                                                              GGT                                                                              CAG                                                                              GAC                                                                              TTG                                                                              GAT                                                                              ATG                              GAA                                                                              AGG                                                                              GAC                                                                              ATA                                                                              ACT                                                                              CCA                                                                              GCA                                                                              CTG                                                                              TCA                                                                              CCG                                                                              TGT                                                                              GTC                                                                              GTC                                                                              AGC                                                                              AGC                                                                              AGT                              CTC                                                                              TCT                                                                              GAG                                                                              TGG                                                                              CAT                                                                              ATG                                                                              CAG                                                                              ATG                                                                              GAC                                                                              ATT                                                                              ATA                                                                              CCA                                                                              GAT                                                                              AGC                                                                              ACC                                                                              ACT                              GAT                                                                              CTG                                                                              TAT                                                                              AAC                                                                              CTA                                                                              CAG                                                                              GTG                                                                              TCA                                                                              CCC                                                                              ATG                                                                              CCT                                                                              TCC                                                                              ACC                                                                              TCC                                                                              GAA                                                                              GCC                              GCA                                                                              ACA                                                                              GAC                                                                              GAG                                                                              GAT                                                                              GAG                                                                              GAA                                                                              GGG                                                                              AAG                                                                              ATA                                                                              GCC                                                                              GAA                                                                              GAC                                                                              CTT                                                                              ATG                                                                              AAG                              CTC                                                                              TTT                                                                              GAA                                                                              CAG                                                                              TCT                                                                              GAG                                                                              TGG                                                                              CAG                                                                              CCG                                                                              ACA                                                                              CAC                                                                              ATC                                                                              GAT                                                                              GGC                                                                              AAG                                                                              GGA                              TAC                                                                              TTG                                                                              CTC                                                                              AAT                                                                              GAG                                                                              CCA                                                                              GGG                                                                              ACC                                                                              CAG                                                                              CTC                                                                              TCT                                                                              TCT                                                                              GTC                                                                              TAT                                                                              GGA                                                                              GAC                              TTC                                                                              AGC                                                                              TGC                                                                              AAA                                                                              GAG                                                                              GAA                                                                              CCA                                                                              GAG                                                                              ATT                                                                              GAC                                                                              AGC                                                                              CCT                                                                              CGA                                                                              GGG                                                                              GAC                                                                              ATT                              GGG                                                                              ATA                                                                              GGC                                                                              ATA                                                                              CAA                                                                              CAT                                                                              GTC                                                                              TTC                                                                              ACG                                                                              GAG                                                                              ATG                                                                              AAG                                                                              AAT                                                                              ATG                                                                              GAC                                                                              TCC                              ATC                                                                              ATG                                                                              TGG                                                                              ATG                                                                              GAC                                                                              AGC                                                                              CTG                                                                              CTG                                                                              GGC                                                                              AAC                                                                              TCT                                                                              GTG                                                                              AGG                                                                              CTG                                                                              CCG                                                                              CCC                              TCT                                                                              ATT                                                                              CAG                                                                              GCC                                                                              ATT                                                                              CCT                                                                              TGT                                                                              GCA                                                                              CCA                                                                              TAG.                                               __________________________________________________________________________


7. The composition of claim 6, wherein said nucleic acid sequencecomprises upstream and downstream sequences that flank the sequenceencoding said IRF-1, and said upstream and downstream sequences and saidIRF-1 encoding sequences comprise ##STR19##
 8. The composition of anyone of claims 1-7, wherein said host cell is a bacterial, yeast ormammalian cell.
 9. The composition of claim 8, wherein said host cell isa bacterial cell.
 10. The composition of claim 9, wherein said bacterialcell is an E. coli cell.
 11. The composition of claim 8, wherein saidhost cell is a yeast cell.
 12. The composition of claim 8, wherein saidhost cell is a mammalian cell.
 13. The composition of claim 12, whereinsaid mammalian cell is a mouse cell.
 14. The composition of claim 12,wherein said mammalian cell is a CHO cell.
 15. Purified human IRF-1having the amino acid sequence ##STR20##
 16. Purified murine IRF-1having the amino acid sequence

    __________________________________________________________________________    Met                                                                              Pro                                                                              Ile                                                                              Thr                                                                              Arg                                                                              Met                                                                              Arg                                                                              Met                                                                              Arg                                                                              Pro                                                                              Trp                                                                              Leu                                                                              Glu                                                                              Met                                                                              Gln                                                                              Ile                              Asn                                                                              Ser                                                                              Asn                                                                              Gln                                                                              Ile                                                                              Pro                                                                              Gly                                                                              Leu                                                                              Ile                                                                              Trp                                                                              Ile                                                                              Asn                                                                              Lys                                                                              Glu                                                                              Glu                                                                              Met                              Ile                                                                              Phe                                                                              Gln                                                                              Ile                                                                              Pro                                                                              Trp                                                                              Lys                                                                              His                                                                              Ala                                                                              Ala                                                                              Lys                                                                              His                                                                              Gly                                                                              Trp                                                                              Asp                                                                              Ile                              Asn                                                                              Lys                                                                              Asp                                                                              Ala                                                                              Cys                                                                              Leu                                                                              Phe                                                                              Arg                                                                              Ser                                                                              Trp                                                                              Ala                                                                              Ile                                                                              His                                                                              Thr                                                                              Gly                                                                              Arg                              Tyr                                                                              Lys                                                                              Ala                                                                              Gly                                                                              Glu                                                                              Lys                                                                              Glu                                                                              Pro                                                                              Asp                                                                              Pro                                                                              Lys                                                                              Thr                                                                              Trp                                                                              Lys                                                                              Ala                                                                              Asn                              Phe                                                                              Arg                                                                              Cys                                                                              Ala                                                                              Met                                                                              Asn                                                                              Ser                                                                              Leu                                                                              Pro                                                                              Asp                                                                              Ale                                                                              Glu                                                                              Glu                                                                              Val                                                                              Lys                                                                              Asp                              Gln                                                                              Ser                                                                              Arg                                                                              Asn                                                                              Lys                                                                              Gly                                                                              Ser                                                                              Ser                                                                              Ala                                                                              Val                                                                              Arg                                                                              Val                                                                              Tyr                                                                              Arg                                                                              Met                                                                              Leu                              Pro                                                                              Pro                                                                              Leu                                                                              Thr                                                                              Arg                                                                              Asn                                                                              Gln                                                                              Arg                                                                              Lys                                                                              Glu                                                                              Arg                                                                              Lys                                                                              Ser                                                                              Lys                                                                              Ser                                                                              Ser                              Arg                                                                              Asp                                                                              Thr                                                                              Lys                                                                              Ser                                                                              Lys                                                                              Thr                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Leu                                                                              Cys                                                                              Gly                                                                              Asp                                                                              Val                                                                              Ser                              Pro                                                                              Asp                                                                              Thr                                                                              Phe                                                                              Ser                                                                              Asp                                                                              Gly                                                                              Leu                                                                              Ser                                                                              Ser                                                                              Ser                                                                              Thr                                                                              Leu                                                                              Pro                                                                              Asp                                                                              Asp                              His                                                                              Ser                                                                              Ser                                                                              Tyr                                                                              Thr                                                                              Thr                                                                              Gln                                                                              Gly                                                                              Tyr                                                                              Leu                                                                              Gly                                                                              Gln                                                                              Asp                                                                              Leu                                                                              Asp                                                                              Met                              Glu                                                                              Arg                                                                              Asp                                                                              Ile                                                                              Thr                                                                              Pro                                                                              Ala                                                                              Leu                                                                              Ser                                                                              Pro                                                                              Cys                                                                              Val                                                                              Val                                                                              Ser                                                                              Ser                                                                              Ser                              Leu                                                                              Ser                                                                              Glu                                                                              Trp                                                                              His                                                                              Met                                                                              Gln                                                                              Met                                                                              Asp                                                                              Ile                                                                              Ile                                                                              Pro                                                                              Asp                                                                              Ser                                                                              Thr                                                                              Thr                              Asp                                                                              Leu                                                                              Tyr                                                                              Asn                                                                              Leu                                                                              GlN                                                                              Val                                                                              Ser                                                                              Pro                                                                              Met                                                                              Pro                                                                              Ser                                                                              Thr                                                                              Ser                                                                              Glu                                                                              Ala                              Ala                                                                              Thr                                                                              Asp                                                                              Glu                                                                              Asp                                                                              Glu                                                                              Glu                                                                              Gly                                                                              Lys                                                                              Ile                                                                              Ala                                                                              Glu                                                                              Asp                                                                              Leu                                                                              Met                                                                              Lys                              Leu                                                                              Phe                                                                              Glu                                                                              Gln                                                                              Ser                                                                              Glu                                                                              Trp                                                                              Gln                                                                              Pro                                                                              Thr                                                                              His                                                                              Ile                                                                              Asp                                                                              Gly                                                                              Lys                                                                              Gly                              Tyr                                                                              Leu                                                                              Leu                                                                              Asn                                                                              Glu                                                                              Pro                                                                              Gly                                                                              Thr                                                                              GlN                                                                              Leu                                                                              Ser                                                                              Ser                                                                              Val                                                                              Tyr                                                                              Gly                                                                              Asp                              Phe                                                                              Ser                                                                              Cys                                                                              Lys                                                                              Glu                                                                              Glu                                                                              Pro                                                                              Glu                                                                              Ile                                                                              Asp                                                                              Ser                                                                              Pro                                                                              Arg                                                                              Gly                                                                              Asp                                                                              Ile                              Gly                                                                              Ile                                                                              Gly                                                                              Ile                                                                              Gln                                                                              His                                                                              Val                                                                              Phe                                                                              Thr                                                                              Glu                                                                              Met                                                                              Lys                                                                              Asn                                                                              Met                                                                              Asp                                                                              Ser                              Ile                                                                              Met                                                                              Trp                                                                              Met                                                                              Asp                                                                              Ser                                                                              Leu                                                                              Leu                                                                              Gly                                                                              Asn                                                                              Ser                                                                              Val                                                                              Arg                                                                              Leu                                                                              Pro                                                                              Pro                              Ser                                                                              Ile                                                                              Gln                                                                              Ala                                                                              Ile                                                                              Pro                                                                              Cys                                                                              Ala                                                                              Pro.                                                  __________________________________________________________________________